Spatial cue utilization in chronically malnourished rats: task-specific learning deficits.

Rats whose mothers were maintained on either a 25% casein diet or an 8% casein diet and who were provided the same diet after weaning were tested on delayed spatial alternation or on one of a series of spatial localization problems using the Morris maze (Morris, 1981). Malnourished rats demonstrated perseverative deficits in the form of strings of consecutive errors on the delayed spatial alternation. Performance in the Morris maze indicated spatial localization ability and spatial memory processes were not impaired by chronic malnutrition in rats. The data suggest that complex processing of spatial information that includes flexible use of place cues over short intervals is impaired by malnutrition, while spatial localization per se and spatial mapping are not affected.

Altered development of responsiveness to clonidine in severely malnourished rats.

To examine the effects of malnutrition on the ontogeny of alpha 2 noradrenergic receptor function, we compared the effects of clonidine during early development in severely malnourished and well-nourished rat pups. Independent groups of pups from dams given either 6% or 25% casein diets received one of five doses of clonidine at 5, 10, 15, 20 or 25 days of age and dose-response relationships for motor activity were determined. In the 25% pups the clonidine-induced locomotor activity was greatest at 5 and 10 days, intermediate at 15 days and not elevated at 20 and 25 days. The malnourished pups exhibited a significant delay in the transition from hyperactivity to hypoactivity, being activated by clonidine until at least 25 days. Wall-climbing measures indicated similar developmental trends as overall activity. These results are discussed in terms of the proposed mechanisms mediating the developmental change in the effects of alpha 2 receptor stimulation.

Generational effects of protein malnutrition in the rat.

The most prevalent form of malnutrition in humans is characterized by its chronic and generational nature. We, therefore, have carried out preliminary studies in rats of the generational effects of protein malnutrition. Our studies to date indicate that a mild protein restriction (8% casein diet) in the first generation becomes a more severe protein restriction in the second generation. This is based on weight gains of the dams during pregnancy, the mean number of pups (F2) per litter, the mean pup (F2) body weight and brain weight at birth, growth curves, levels of brain tryptophan (TRP), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) from birth to weaning, and the levels of certain plasma constituents, especially non-esterified fatty acids (NEFA). We propose this paradigm as an animal model for some types of chronic undernutrition in socioeconomically underprivileged human populations.

Developmental protein malnutrition in the rat: effects on single-unit activity in the frontal cortex.

This study evaluated the effects of developmental protein malnutrition on the spontaneous electrical activity of frontal cortex neurons in the anesthetized rat. Rats were raised prenatally and postnatally on either an 8% or 6% casein diet until adulthood. Compared to the 25% casein controls, both malnourished groups showed a 30-36% decrease in mean discharge rates and a 100-200% increase in the percentage of cells with very slow (less than 1/s) discharge rates. Most of the diet-related changes were confined to a zone 600-1200 micron below the brain surface, approximately cortical layers III, IV and V. A second set of studies in which diet reversals were introduced at birth or in adulthood found that: (a) restoration of a normal 25% casein diet at birth did not appreciably attenuate the effect of prenatal administration of an 8% casein diet; (b) introduction in adulthood of the 8% casein diet to a normally fed rat had no effect; (c) introduction of the 8% diet at birth, however, produced effects in adulthood comparable to those seen when the protein malnutrition was introduced in the prenatal period. Thus, the rat brain is sensitive to both prenatal and postnatal protein malnutrition (starting at birth). Most importantly, the effects of prenatal protein malnutrition on the activity of frontal cortex neurons do not appear to be reversible by restoration of a normal diet in adulthood or at birth.

Ontogeny of the levels of serotonin in various parts of the brain in severely protein malnourished rats.

We have previously reported that irreversible central and peripheral chemical changes and irreversible changes in spontaneous activity of single neurons in the frontal cortex are seen in adult rats who were born to mothers fed an 8% isocaloric casein diet 5 weeks before mating and during gestation, who were cross-fostered at birth by control (25% casein diet fed) dams and who showed a normal growth curve. Thus, in the rat, a normal growth curve does not necessarily mean a normal development. We now report similar irreversible, albeit larger, changes in small-for-gestational-age (SGA) rats born to mothers fed a 6% isocaloric diet, nursed by 6% casein fed dams and who showed an abnormal growth curve. The regional ontogeny of serotonin, tryptophan and 5-HIAA in the brain and plasma levels of tryptophan, protein, albumin and NEFA are reported. Thus, similar changes were observed in animals with a normal growth curve and in animals with a very markedly decreased growth curve resembling marasmus in humans.

The effects of protein deprivation on the nucleus locus coeruleus: a morphometric Golgi study in rats of three age groups.

In a previous morphometric Golgi study of the nucleus locus coeruleus we identified in rats fed a 25% casein diet 3 cell types, fusiform, multipolar and ovoid, and compared their age-related changes from 30 to 90 days and 90 to 220 days. In the present study we investigated the effects of an 8% casein diet, initiated prenatally and continued postnatally in the pups, using the same morphometric parameters at the same 3 ages. In these rats the majority of significant age-related changes were in primary and secondary dendritic spine density. On all 3 cell types between 30 and 90 days there was a decreased spine density followed by an increase between 90 and 220 days. These age-related changes closely followed those in controls and, as a result, when the two diet groups were compared at each age they showed only 4 significant differences out of 18 comparisons. In control rats these age-related changes in dendritic spine density in the nucleus locus coeruleus were diametrically out-of-phase with those found in the nucleus raphe dorsalis in a previous study. This is consistent with the postulated mutual inhibitory relationship between the nucleus locus coeruleus and nucleus raphe dorsalis. Comparison of these same events in the nucleus locus coeruleus and nucleus raphe dorsalis in the 8% casein diet rats show no evidence of an out-of-phase relationship. Thus, the relationship between these two closely related nuclei appears to be fundamentally altered by the 8% casein diet. In these comparisons of dendrite spine density and other parameters the 8% casein diet rats have shown in both the nucleus locus coeruleus and nucleus raphe dorsalis deficits and increases as compared to controls. In contrast, in morphometric Golgi studies of pyramidal and Purkinje cells undernutrition generally results in deficits or little change in the various parameters. This suggests that the adaptation of the non-pyramidal neurons in the present study is different from that shown by pyramidal and Purkinje cells.

Animal models for small-for-gestational-age (SGA) neonates and infants-at-risk (IAR).

In this study, the body weight changes seen in rat dams and brain and body weights seen in pups as sequelae of either an overt or a hidden form of chronic protein deprivation have been examined. In the overt model, the 6% casein-diet pups showed SGA brain and body weight deficits and irreversible central and peripheral chemical changes at birth. In contrast, the hidden form of prenatal deprivation did not result in brain and body weight deficits in the 8% casein-diet pups at birth, but their SGA-like irreversible peripheral and central chemical profiles categorizes them as IAR-neonates. In cross-fostering experiments it was shown that pups from dams fed an 8% casein-diet cross-fostered onto 25% casein-diet dams at birth showed no significant differences in brain or body weight at weaning, whereas pups from 6% casein-diet dams cross-fostered onto 25% casein-diet dams at birth continued to show significantly lower brain and body weights at weaning. These studies show that the 8% casein-diet rat at birth is an IAR model because of its irreversible peripheral and central chemical changes dating form birth, even though the body and brain weights are essentially the same as the pups from 25% casein-diet dams. On the other hand, the pups from 6% casein-diet dams are SGA animals at birth and these brain and body weight deficits at birth cannot be completely reversed at weaning even when the pup is cross-fostered to a 25% casein-diet dam at birth.(ABSTRACT TRUNCATED AT 250 WORDS)

Overt and hidden forms of chronic malnutrition in the rat and their relevance to man.

We have examined the physiological weight changes seen in rat dams and their offspring as sequelae of either an overt or a hidden form of chronic protein malnutrition. In the overt model, which was produced by feeding dams a very low protein diet (6% casein) starting 5 weeks prior to conception and continued through lactation, the females showed significant weight losses at all ages compared to dams maintained on a normal diet (25% casein). This caused the malnourished 6% dams to have offspring that were categorized as small-for-date at birth in terms of their weight indices and peripheral metabolic profiles. Also, the inadequate milk production of these dams resulted in their pups displaying the almost total failure of growth (greater than 60% decreases in body weights) and peripheral imbalances characteristic of infantile marasmus by day 8 of lactation. Consequently, at all times examined the 6% dams and pups showed most of the typical responses seen in the more severe forms of in utero and lactational malnutrition of mankind. In contrast, the hidden form of malnutrition produced by feeding dams a somewhat higher protein diet (8% casein) throughout the same time periods caused no marked weight losses by these females during their pregnancy compared to the normal dams. Although the 8% pups had the same birth weight indices as the normal offspring, previous data from our group have indicated that the 8% progeny show many metabolic imbalances at birth which are indicators of severe gestational malnutrition in humans. Moreover, while the 8% dams displayed lactational insufficiencies as noted by their pups retarded postnatal growth, nursing of these offspring by 25% dams allowed them to maintain a normal lactational growth curve. However, not only was this cross-fostering unable to rehabilitate most of the prenatally determined biochemical alterations affecting the 8% pups but, additionally, this form of malnutrition will remain undetected if weight indices alone are used as assessors of normalcy. Thus, it appears that the 8% rats may serve as a useful model for the hidden forms of malnutrition in man.

The effects of protein deprivation on the nucleus raphe dorsalis: a morphometric Golgi study in rats of three age groups.

In a previous study we identified 3 cell types in the nucleus raphe dorsalis (NRD): fusiform, multipolar and ovoid. In the present study, we have investigated the effect of an 8% casein diet on these 3 cell types using quantitative techniques on rapid Golgi-impregnated neurons from rats of 3 different ages: 30, 90 and 220 days. Major and minor axes of the cell body and dendritic diameter were unaffected and primary dendritic linear extent was only slightly affected by the diet. All 3 cell types in control rats showed an increase in synaptic spines on both primary and secondary dendrites between 30 and 90 days followed by a decrease for all 3 of the cell types at 220 days. Protein-deprived rats failed to show these age-related changes. Other parameters of comparison showed clear differences between the 3 cell types. These differences could be readily seen when total synaptic spine input to the primary and secondary dendrites was calculated from the data on dendritic number, linear extent and spine density. When viewed in this way the fusiform and ovoid cells show either little change or a decreased synaptic input at all ages, while the presumed serotonergic multipolar cells showed an increase. This is in agreement with neurochemical studies in these rats showing increased levels of this biogenic amine in protein malnourished rats.

Nucleus raphe dorsalis: a morphometric Golgi study in rats of three age groups.

Using Rapid Golgi and Nissl techniques, three major cell types: fusiform, multipolar and ovoid-shaped were identified in the nucleus raphe dorsalis of male rats at 30, 90, and 220 days of age. We have described the orientation and dendritic architecture of raphe cells as to type and the relationships of these cells to blood vessels and surrounding structures. For each cell type, the origin of the axon is characteristic. One hundred neurons per age group were measured at their maximal linear extent and the number of spines on the somal surface was counted. Dendritic number, linear extent, diameter and the number of spines along a 50 micron segment near the mid-point of dendritic length in an equal number of primary and secondary dendrites were quantified in each age group. The most striking age-related changes in the multipolar and ovoid-shaped cells were dendritic number, diameter and spine number as well as the number of perisomatic spines. The fusiform cells showed the least age-related changes. In general, the nucleus raphe dorsalis is organized as a reticular nucleus with neurons having few, straight and poorly ramified dendrites.

Adrenalectomy: its effects on systemic tryptophan metabolism in normal and protein malnourished rats.

Rats born to dams fed either a low protein diet (8% casein) or a normal diet (25% casein) started 5 weeks prior to conception and continued through lactation were bilaterally adrenalectomized or received a sham-operation at 30 days of age. At 60 days of age, the systemic tryptophan metabolism of th 8% and 25% adrenalectomized rats was compared to the sham-operated controls of each diet group. While adrenal ablation produced significant decreases in the brain serotonin and metabolite concentrations and marked increases in brain tryptophan concentrations for both diet groups compared to their respective controls, these substances remained significant higher in all malnourished rats than in the well-nourished groups. Also, the major modulator of the peripheral metabolic pathways which regulates the availability of free plasma tryptophan (total tryptophan, albumin, and nonesterified fatty acid concentrations) was the nutritional status of the rats rather than their treatment condition. Only plasma corticosterone concentrations showed changes (significantly decreases) as a consequence of adrenal ablation for either diet group. Overall, the data indicated that under physiological conditions the adrenal cortex has an important function in determining brain tryptophan utilization, whereas its role in regulating peripheral tryptophan metabolism is minimal.

Power spectral analysis of the EEG following protein malnutrition.

In these studies, power spectral analysis techniques were utilized to quantify the EEG obtained from rats reared on either an 8% or 25% casein diet during various vigilance states at two stages of development: (1) adulthood-90 to 120 days old; and (2) immediately after weaning-22 to 23 days old. It was found that the cortical EEG contained relatively more power in the low frequencies (ie., 0.5 to 10 Hz) for the 22-23 day old animals than for the 90-120 day old rats, especially during the slow wave sleep states-SWS1 and SWS2. Theta activity (5-8 Hz) in the hippocampus was shown to have greater power for the 22-23 day old group than for the older animals during both REM sleep and waking. Analyses of power spectral data and other indices of the frequency distribution of the hippocampal EEG indicated that those animals subjected to protein malnutrition have significantly more power in the theta band during REM sleep than the normal adult group. Since it was also noted that the hippocampal EEG obtained from the 22-23 day old group contained relatively more power in the theta band than the 90-120 day old group, the dietary treatment effect might be intrepreted as an instance of retarded development associated with protein malnutrition. Thus, a significant effect of the dietary manipulation used in the study may be largely on the system responsible for regulating theta activity.

Developmental protein malnutrition: influences on the central nervous system of the rat.

Our group has been carrying out interdisciplinary studies on the effects of prenatal and postnatal protein malnutrition on the developing rat brain. Anatomical, physiological, biochemical and behavioral approaches using the same animal model have revealed that protein malnutrition affects the brain at various levels, i.e., (1) anatomical, as revealed by Golgi findings of deranged dendritic trees on analysis of cortical and subcortical areas; (2) physiological, as revealed by delayed sleep pattern maturation, disturbances in seizure thresholds, slowing of sensory cortico-cortical and thalamocortical evoked potentials, and changed power in hippocampal theta activity; (3) biochemical, as revealed by marked increases in biogenic amines dating from birth, as well as modifications in tryptophan metabolism; and (4) behavioral, as revealed by various changes in responses to different kinds of aversive stimulation. Reversal studies have revealed that many changes are permanent and not amenable to nutritional rehabilitation even at birth, which is before the brain growth spurt in the rat. Our paradigm closely mimicks the human condition of low level, chronic protein undernutrition and thus reveals the underlying disturbances due to malnutrition. The dietary reversal studies are attempts at pin-pointing critical brain growth periods, beyond which recovery of functions is not possible.

Utilization of 14C-tyrosine in brain and peripheral tissues of developmentally protein malnourished rats.

Prior studies of developmentally protein malnourished rats have reported substantial changes in brain and peripheral utilization of 14C-leucine, 14C-phenylalanine, and 14C-tryptophan. In the present study rats born to dams fed a low protein diet (8% casein) compared to the offspring of control rats fed a normal diet (25% casein) showed few significant differences in the uptake and incorporation of 14C-tyrosine into brain and peripheral tissues from birth to age 21 days. At birth, the 8% casein pups exhibited significant decreases in brain and peripheral tissue incorporation of tracer only at short post-injection times (10 and 20 min), but not at longer intervals (90 and 180 min). During ontogenetic development (Days 5-21), the 8% casein rats showed significant increases in uptake of 14C-tyrosine into the brain and peripheral tissues on Day 11 and a significantly higher percent incorporation of tracer into brain protein on Day 21 as compared to the 25% casein rats. For the most part, there were no significant changes in incorporation of radioactivity in peripheral tissues for the 2 diet groups on these post-birth days. Overall, the data indicates that developmental protein malnutrition causes relatively fewer changes in brain and peripheral utilization of the semi-essential amino acid tyrosine than those observed in previous studies with essential amino acids.

A neuropharmacological analysis of central nervous system catecholamine systems in development protein malnutrition.

Four experiments were performed to evaluate the effects of developmental protein deprivation on the behavioral response of adult rats to treatments known to affect central nervous system catecholamine systems. Results showed no group differences between protein malnourished and control animals in locomotor responsiveness to d- or l-amphetamine, recovery from behavioral asymmetry produced by a unilateral lesion of the substantia nigra, or in the development of response patterns indicative of denervation supersensitivity. However, a dose-dependent diminution in the ability of apomorphine to produce stereotyped behavior was noted in the malnourished group, suggesting that a class of brain dopamine receptors may be impaired or may have undergone homeostatic modification as a result of the undernutrition procedure.